The Role Of Amhy/Amh And Their Receptor Amhrâ…¡ In Nile Tilapia Male Sex Determination

Master sex-determining (SD) genes are the key genetic switches controlling the gonadal sex differentiation cascade leading to the development of either ovaries or testes. Amh and AmhrⅡ belong to members of TGF-(3 superfamily. Amh and AmhrⅡ are responsible for the regression of Mullerian ducts in tetrapods. Amh and AmhrⅡ are also found in teleost fish despite the fact that they do not have Miillerian ducts. Variation in the TGF-β signaling pathway is emerging as an important mechanism by which gonadal sex determination is controlled in teleosts. These include a Y-linked duplicate of the anti-Mullerian hormone (Amhy) in the Patagonian pejerrey, a mutation in the amh receptor (AmhrⅡ) in Takifugu rubripes, and a Y-linked duplicate of a related ligand, gonadal soma derived growth factor (gsdfY) in Oryzias luzonensis. The Nile tilapia (Oreochromis niloticus), a gonochoristic teleost with a stable XX/XY sex determination system, has become one of the most important species in global aquaculture. Moreover, it is a good experimental model for understanding the developmental genetic basis of sex determination because of the availability of monosex populations and the whole genome sequence.With the help of X-or Y-specific molecular markers and the microarrayed fosmid library constructed by our group, the candidate sex-determining gene Amhy/Amh (sex-determining gene on Y chromosome) of the Nile tilapia was identified by positional cloning in our previous studies. In the present study, the function of Amhy/ Amh was studied by transgenic overexpression of Amhy/Amh in XX and knockout of AmhylAmh in XY by CRISPR/Cas9. At the same time, knockout of Amh receptor AmhrⅡ in XY by CRISPR/Cas9 was also performed. Therefore, the function of Amhyl Amh in tilapia sex determination was elucidated by gain of funtion and loss of funtion. The main results are as follows:1) Disruption of Amhy/Amh in XY tilapia through CRISPR/Cas9 system was achieved with high efficiency (up to 90%) and average of 68%. Subcloning and sequencing confirmed that targeted loci had different mutation types. Furthermore, obvious phenotypes were observed in GO generation after mutation of Amhy/Amh. Histological examination showed that Amhy/Amh deficient gonads of from XY fish exhibited complete sex reversal, while gonads from XY control developed as testes. Immunohistochemistry (IHC) analysis indicated that Cypl9ala (aromatase, the key enzyme responsible for the production of the 17β-estradiol), which was only expressed in interstital cells of the ovary, was found to be expressed in Amhy/Amh deficient ovary. Consistently, Dmrtl, a Sertoli cell marker, was detected in the XY testes but not in the Amhy/Amh deficient gonads. In addition, blood samples from the Amhy/Amh deficient and control fish were collected and serum E2 was measured by euzyme-linked immunosorbent assay (EIA). Consistent with the results from IHC, knockout of Amhy/Amh in XY fish resulted in significantly increased level of E2, compared with the control. Sperm from the XY founders carrying Amhy/Amh mutations was then used to produce F1 animals. In the F1 fish, the gonads of heterozygous XY with single mutantion of Amhy or Amh allele on the Y chromosome displayed no sex reversal with typical testicular structure. In contrast, XY fish with Amhy and Amh double mutation on Y chromosome had a clear ovarian structure characterized by the presence of ovarian cavity (OC) and the appearance of phase Ⅱ oocytes. Furthermore, we used transgenic technique to overexpress Amhy/Amh in XX fish. Histological examination showed that overexpression of Amhy/Amh in XX fish, using a fosmid transgene that carries the Y-linked Amhy/Amh haplotype, resulted in female to male sex reversal with all kinds of spermatogenic cells. By IHC Dmrtl was found to be expressed in the somatic cells of the sex reversal XX testes but not in the control ovary.2) AmhrⅡ was isolated from tilapia genome and tancriptome sequences. AmhrⅡ open reading frames is 1515bp, encoding 504 amino acids. Sequence analysis showed that AmhrⅡ was relatively conserved in the fish. Synteny analysis revealed that conserved synteny of AmhrⅡ and its neighbouring genes was only found between tilapia and medaka not in fugu and tetrapods. Tissue distribution analysis of AmhrⅡ by RT-PCR and real-time PCR showed that AmhrⅡ was found to be mainly expressed in the testis and ovary of the adult fish, with higher expression in the testis. The expression profile of AmhrⅡ in gonads during the development was studied by transcriptome analysis. AmhrⅡ was expressed at very low levels in gonads of both sexes at 5 dah, peaked in gonads of both sexes at 30 dah, with much higher expression in the testis than in the ovary. Thereafter, AmhrⅡ began to decrease, and maintained at intermediate levels till 180 dah.3) Disruption of AmhrⅡ in XY tilapia through CRISPR/Cas9 system was achieved with high efficiency (up to 70%) and average of 48%. Subcloning and sequencing confirmed that the targeted locus had different mutation types. Furthermore, obvious phenotypes were observed in GO generation after mutation of AmhrⅡ. Compared with XY control, histological examination showed that 10,30 and 90 dah AmhrⅡ deficiency XY gonad exhibited complelte sex reversal, with phase Ⅰ and Ⅱ oocytes and the ovarian cavity in the XY fish. IHC analysis revealed that Cyp19a1a was found to be expressed while Dmrt1 not in the AmhrⅡ deficiency XY ovary. In addition, blood samples from the AmhrⅡ deficiency and control fish were collected and serum E2 was measured by EIA. Consistent with the results from IHC and real-time PCR, AmhrⅡ deficiency in the XY fish resulted in significantly increased level of E2, higher than the XY control but lower than the XX control.In summary, Simultaneous knockout of Amhy and Amh in XY fish resulted in male to female sex reversal, while mutation of Amhy or Amh alone did not. In contrast, overexpression of Amhy/Amh in XX fish, using a fosmid transgene, resulted in female to male sex reversal, while overexpression of in XX fish resulted in no sex reversal. In addition, knockout of the AmhrⅡ in XY fish also resulted in complete male to female sex reversal. Taken together, these results strongly suggested that both Amhy and Amh on the Y chromosome are essential for male sex determination in tilapia. The role of the TGF-β signaling pathway is conserved in fish sex determination.